Summary Many flaviviruses are significant human pathogens, among which dengue virus is the most important mosquito-borne viral pathogen with over 390 million infections per year. Apart from dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and recently Zika virus cause global outbreaks and epidemics, posing constant threats to public health. No FDA-approved antiviral drug is currently available to treat any flavivirus infection, and vaccines are lacking against several family members. Thus, the development of flavivirus antiviral therapy is a public health priority. The goal of this proposal is to develop direct antiviral agents (DAAs) for flavivirus therapy. We will pursue three Specific Aims to develop flavivirus DAAs. Aim 1. Develop preclinical DAAs targeting flavivirus capsid and NS4B protein. Aim 2. Identify novel Nuc DAAs with pan-flavivirus activity. Aim 3. Evaluate combination therapy directed against flavivirus infection. We will deliver the proposed milestones by combining the cutting-edge biology from academia and drug discovery acumen from industry with clear go/no-go criteria.